US20240016925A1

US20240016925A1 - Novel vaccine adjuvant

Info

Publication number: US20240016925A1
Application number: US18/365,671
Authority: US
Inventors: Yong Bok Seo; Youngwoo Choi; Sang Hee SIM
Original assignee: Sl Vaxigen Inc
Current assignee: Sl Vaxigen Inc
Priority date: 2018-02-02
Filing date: 2023-08-04
Publication date: 2024-01-18
Also published as: EP3747459A4; CN111918666A; TW201938793A; US20200360514A1; JP2021512950A; WO2019151759A1; US11844834B2; EP3747459A1; KR102286397B1; KR20190094107A

Abstract

There is provided a novel vaccine adjuvant, and more specifically, a vaccine adjuvant for stimulating a T lymphocyte-specific immune response, which includes an IL-12 protein and an IL-21 protein as active ingredients, or includes the polynucleotide encoding an IL-12 protein and the polynucleotide encoding an IL-21 protein as active ingredients.

Description

TECHNICAL FIELD

This application claims priority to Korean Patent Application No. 2018-0013329 filed Feb. 2, 2018. The specification of the above application is incorporated herein by reference in its entirety.
The present invention relates to a vaccine adjuvant, and more specifically, to a novel vaccine adjuvant that can maximize the therapeutic effect of anticancer vaccines, etc. by selectively enhancing a T cell-specific immune response.

BACKGROUND

Vaccines are drugs used to generate an immune response to antigens for the defense against pathogenic infections and recently developed vaccines mainly use recombinant proteins as antigens. Recombinant proteins are safe and less toxic compared to killed vaccines or attenuated live vaccines, but the recombinant proteins have low immunogenicity. Therefore, vaccine adjuvants are used together so as to provide sufficient immunity to defend against infection.
Vaccine adjuvants are classified into three types, i.e., a carrier of an antigen, an immunostimulant, and one which acts as a matrix for an antigen while stimulating an immune response according to the mechanism of action. The effective use of vaccine adjuvants can allow to obtain various effects, such as: (1) increase the immunogenicity of recombinant antigens, (2) reduce the dose of antigens or reduce the number of immunizations, and (3) improve immunogenicity in infants and elderly people with weak immunity. However, despite the usefulness of these vaccine adjuvants, there are still few vaccine adjuvants licensed for use in human vaccines, and this is because there are a number of things to consider in order to be selected as a vaccine adjuvant. The ideal conditions for vaccine adjuvants are that they should be safe, excellent in drug resistance, simple to manufacture, and low in production cost. Additionally, vaccine adjuvants should have a long half-life in vivo and be biodegradable, and should not induce any immune response against the immune adjuvants themselves. In particular, among them, the safety issue of vaccine adjuvant is most important.
The vaccine adjuvants currently approved in Europe and USA for use in vaccines include an aluminum salt, MF59, ASO3, ASO4, etc. Aluminum salts are mainly used in the form of Al(OH)₃or AlPO₄, which are thought to exhibit an immunostimulating effect by adsorbing to and slowly releasing protein antigens. Recently, however, aluminum salts have been shown to activate dendritic cells and promote secretion of cytokines (e.g., IL-10 and IL-18). Aluminum salts are widely used in several vaccines and are thought to be very safe, but it is assumed that aluminum salts may cause allergic reactions and have neurotoxicity. In addition, aluminum salts strongly induce antibody-mediated humoral immune responses, but aluminum salts have disadvantages in that they hardly induce cell-mediated immune responses and cryopreservation is not possible.
The MF59 (Ott et al., Pharm Biotechnol. 6: 277-296, 1995) of Novartis and the ASO3 (Li et al., J. Virol. 80(3):1414-1426, 2006) of GSK are both immune adjuvants in the form of an oil-in-water (o/w) emulsion with squalene as the basic ingredient. The o/w emulsion immune adjuvant activates antigen-presenting cells to stimulate antigen uptake and cytokine secretion, increases expression of chemokine receptors, thereby increasing migration of antigen-presenting cells. MF59 and ASO3 were used for the purpose of increasing the immunogenicity of the antigens in the vaccine against the new type of influenza virus pandemic in 2009 and reducing the amount of antigen used, through clinical trials, MF59 and ASO3 were confirmed to have effects of increasing the rate of antibody production and cross-defense effect. In addition to squalene, various emulsion immune adjuvants have been developed using emulsions prepared in nature that can be used in humans, but the commercialization of these emulsion immune adjuvants is difficult due to excessive immune responses and toxicities. Particulate carriers such as liposomes, immunostimulatory complexes, and virus-like particles are also being developed as immune adjuvants, and these are usually developed as vaccine carriers and adjuvants by encapsulating the antigens. Particularly, liposomes have been actively studied. Liposomes are synthetic spheres constituting a lipid layer that can encapsulate antigens and simultaneously act as a vaccine delivery and immune adjuvant, and the activity of liposomes varies depending on the number, charge, composition, and preparation method of the lipid layer. Liposomes enhance humoral immunity and cellular immunity against protein and polysaccharide antigens. However, the use of liposomes is limited due to difficulties in preparation, stability, etc., and it is necessary to add an immunostimulatory component in order to have strong actions of immune activity, especially because the role of the immunostimulatory component is closer to an antigenic carrier than an adjuvant to enhance immune response. Currently, GSK has developed an immune adjuvant, ASO1B, in which monophosphoryl lipid A (MPL) is added to liposomes, and is now attempting to develop the immune adjuvant to be applicable as a vaccine adjuvant for patients with tuberculosis and malaria diseases (WO96/33739A).
However, although these vaccine adjuvants enhance the cell-mediated immune response, most of these vaccine adjuvants have been developed to enhance the immune response of the vaccine composition for the prevention of infectious diseases (e.g., viruses, etc.), which require mainly humoral immunity, and thus the effects of vaccines for the prevention and treatment of diseases that require cell-mediated immune responses (e.g., cancer, etc.) have not yet been confirmed.

SUMMARY

As described above, there is still an urgent need for the development of a vaccine adjuvant, by enhancing T-cell specific immune responses, which is effective for the prevention and treatment of diseases that require enhancing cell-mediated immune responses (e.g., cancer, etc.) as well as the prevention and treatment of conventional infectious disease caused by the infection of viruses, etc. In this regard, the present inventors, to solve the various issues including those described above, provides a novel vaccine adjuvant that can enhance the effects of the vaccine via the enhancement of T cell immune response-specific immunity. However, the present invention is not limited by the purpose.
In an aspect of the present invention, there is provided a vaccine adjuvant for stimulating a T lymphocyte-specific immune response, in which the vaccine adjuvant includes a polynucleotide encoding an IL-12 protein and a polynucleotide encoding an IL-21 protein as active ingredients.
In another aspect of the present invention, there is provided a vaccine adjuvant for stimulating a T lymphocyte-specific immune response, in which the vaccine adjuvant includes an IL-12 protein and an IL-21 protein as active ingredients.
According to still another aspect of the present disclosure, there is provided a vaccine composition for stimulating a T lymphocyte-specific immune response, in which the vaccine composition includes the vaccine adjuvant and an antigen for immunization as active ingredients.
According to still another aspect of the present disclosure, there is provided a method for stimulating a T lymphocyte-specific immune response by the vaccine composition, in which the method includes administering, to an individual, the vaccine adjuvant along with a vaccine composition, or before or after the administration of the vaccine composition.

Effect of the Invention

The vaccine adjuvant according to an embodiment of the present invention significantly enhances the T cell-specific immune response by selectively increasing T cells that specifically respond to antigens without affecting antibody production. Thus, the vaccine adjuvant can be usefully used as an immunotherapeutic agent for treating cancer such as cervical cancer as well as for treating infectious diseases by the infection of various infectious viruses through intensifying the cellular immune.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments can be understood in more detail from the following description taken in conjunction with the accompanying drawings, in which:

FIG. 1 a is a schematic diagram showing the gene construct of BD-121, which is a vaccine adjuvant in accordance with an exemplary embodiment; and FIG. 1 b is a schematic diagram showing the gene construct of BD-121A, which is a vaccine adjuvant in accordance with an exemplary embodiment;

FIG. 2 is schematic diagrams showing the gene construct of BD-14, which is a 14-valent HPV DNA vaccine prepared in accordance with an exemplary embodiment;

FIG. 3 is a schematic diagram showing the gene construct of a CMV DNA vaccine, which is prepared in accordance with an exemplary embodiment;

FIG. 4 a is a schematic diagram showing the gene construct of BD-02B, which is included in a HSV-2 DNA vaccine prepared in accordance with an exemplary embodiment; and FIG. 4 b is a schematic diagram showing the gene construct of BD-02C, which is included in a HSV-2 DNA vaccine prepared in accordance with an exemplary embodiment;

FIG. 5 is a schematic diagram showing the gene construct of an SFTS DNA vaccine, which is prepared in accordance with an exemplary embodiment;

FIG. 6 is a graph showing the results of ELISPOT analysis of antigen-specific T cell immune responses when the conventional 2-valent HPV DNA vaccine is administered alone or in combination with the vaccine adjuvant BD-121 in accordance with an exemplary embodiment;

FIG. 7 a is graphs showing the results of ELISA assay of concentrations of IL-12 and IL-21 in the culture supernatant of COS-7 cells transfected with a hBD-121 construct, which is a vaccine adjuvant in accordance with an exemplary embodiment;

FIG. 7 b is graphs showing the results of ELISA assay of concentrations of IL-12 and IL-21 in the culture supernatant of COS-7 cells transfected with an mBD-121 construct, which is a vaccine adjuvant in accordance with an exemplary embodiment;

FIG. 7 c shows the results of western blot analysis of expression levels of IL-12 and IL-21 in the culture supernatant of COS-7 cells transfected with a hBD-121 construct and an mBD-121 construct, respectively, which are vaccine adjuvants in accordance with an exemplary embodiment;

FIG. 8 a shows the administration schedule for a CMV DNA vaccine prepared in accordance with an exemplary embodiment and BD-121, which is a vaccine adjuvant prepared in accordance with an exemplary embodiment;

FIG. 8 b is a graph showing the results of counting the number of spleen cells which responds in a CMV antigen-specific manner when a CMV DNA vaccine prepared in accordance with an exemplary embodiment is administered alone or in combination with IL-12, IL-21, or BD-121, which is a vaccine adjuvant prepared in accordance with an exemplary embodiment;

FIG. 8 c is a graph showing the results of analysis of antigen-specific antibody responses when a CMV DNA vaccine prepared in accordance with an exemplary embodimentis administered alone or in combination with IL-12, IL-21, or BD-121, which is a vaccine adjuvant prepared in accordance with an exemplary embodiment;

FIG. 9 a is a graph showing the results of counting the number of spleen cells which responds in a HBV antigen (HBsAg, HBcAg, and PreS1/S2)-specific manner when a HBV DNA vaccine prepared in accordance with an exemplary embodiment is administered alone or in combination with BD-121, which is a vaccine adjuvant prepared in accordance with an exemplary embodiment;

FIG. 9 b is a graph showing the results of analysis of HBsAg-specific antibody responses when a HBV DNA vaccine prepared in accordance with an exemplary embodiment is administered alone or in combination with BD-121, which is a vaccine adjuvant prepared in accordance with an exemplary embodiment;

FIG. 10 a shows the administration schedule for an SFTS DNA vaccine prepared in accordance with an exemplary embodiment and BD-121, which is a vaccine adjuvant prepared in accordance with an exemplary embodiment;

FIG. 10 b is a graph showing the results of counting the number of spleen cells which responds in an SFTS antigen (GnGc, NP, and NS)-specific manner when an SFTS DNA vaccine prepared in accordance with an exemplary embodiment is administered alone or once in combination with BD-121, which is a vaccine adjuvant prepared in accordance with an exemplary embodiment (priming);

FIG. 10 c is a graph showing the results of counting the number of spleen cells which responds in an SFTS antigen (GnGc, NP, and NS)-specific manner when an SFTS DNA vaccine prepared in accordance with an exemplary embodiment is administered alone or twice in combination with BD-121, which is a vaccine adjuvant prepared in accordance with an exemplary embodiment (priming and boosting);

FIG. 11 a is a graph showing the results of counting the number of spleen cells which responds in an SFTS antigen (GnGc, NP, and NS)-specific manner when an SFTS DNA vaccine prepared in accordance with an exemplary embodiment is administered alone or once in combination with BD-121, by varying a mixing ratio, which is a vaccine adjuvant prepared in accordance with an exemplary embodiment (priming);

FIG. 11 b is a graph showing the results of counting the number of spleen cells which responds in an SFTS antigen (GnGc, NP, and NS)-specific manner when an SFTS DNA vaccine prepared in accordance with an exemplary embodiment is administered alone or twice in combination with BD-121, by varying a mixing ratio, which is a vaccine adjuvant prepared in accordance with an exemplary embodiment (priming and boosting);

FIG. 12 a shows the administration schedule for a HSV-2 DNA vaccine (BD02) prepared in accordance with an exemplary embodiment and BD-121, which is a vaccine adjuvant prepared in accordance with an exemplary embodiment;

FIG. 12 b is a graph showing the changes in the pathological index of HSV-2 over time, in a group administered with a HSV-2 DNA vaccine (BD02) prepared in accordance with an exemplary embodiment alone (BD-02B+BD-02C) and a group administered in combination with BD-121, which is a vaccine adjuvant prepared in accordance with an exemplary embodiment (BD02 DP);

FIG. 12 c is graphs showing the changes in the survival rate of experimental animals over time, in a group administered with a HSV-2 DNA vaccine (BD02) prepared in accordance with an exemplary embodiment alone (BD-02B+BD-02C) and a group administered in combination with BD-121, which is a vaccine adjuvant prepared in accordance with an exemplary embodiment (BD02 DP);

FIG. 13 a is a schematic diagram showing the administration schedule for experimental animals, in which a HSV-2 DNA vaccine (BD02) prepared in accordance with an exemplary embodiment and BD-121, which is a vaccine adjuvant prepared in accordance with an exemplary embodiment (BD02 DP), are administered by adjusting the doses;

FIG. 13 b is a graph showing the results of recording the number of spleen cells which responds in a HSV-2 antigen (gD, UL39, and ICP4)-specific manner according to the dose of a vaccine adjuvant in experimental animals, where the experimental animals are subjected to primary inoculation (priming) with a HSV-2 DNA vaccine (BD02) prepared in accordance with an exemplary embodiment and BD-121, which is a vaccine adjuvant prepared in accordance with an exemplary embodiment (BD02 DP), by adjusting the doses;

FIG. 14 is a graph showing the results of analysis of the effect of improving T cell-specific immune responses by BD-121, which is a vaccine adjuvant in accordance with an exemplary embodiment, for a conventional 2-valent HPV DNA vaccine, obtained by counting the number of spleen cells which responds in an antigen-specific manner;

FIG. 15 a shows the administration schedule for a 14-valent HPV DNA vaccine prepared in accordance with an exemplary embodiment and BD-121 or BD-121A, which is a vaccine adjuvant prepared in accordance with an exemplary embodiment;

FIG. 15 b is a graph showing the results of analysis of the effect of improving T cell-specific immune responses by BD-121 and BD-121A, which are vaccine adjuvants in accordance with an exemplary embodiment, for a 14-valent HPV DNA vaccine prepared in accordance with an exemplary embodiment, obtained by counting the number of spleen cells which responds in an antigen-specific manner;

FIG. 16 a shows the administration schedule in preclinical experiments for the analysis of the effect of a HSV-2 DNA vaccine (BD-02) in accordance with an exemplary embodiment and BD-121A, which is a vaccine adjuvant in accordance with an exemplary embodiment in cynomolgus monkeys, which are experimental animals; and

FIG. 16 b is graphs showing the results of counting the number of spleen cells which responds in a HSV-2 antigen (gD2 and UL39)-specific manner in a group where BD-02 is administered alone (left) without a vaccine adjuvant in accordance with an exemplary embodiment and a group where BD-02 is administered in combination with BD-121A (right), pre-administration (VS), one administration (VT1), and two administrations (VT2).

DETAILED DESCRIPTION OF THE INVENTION

In an aspect of the present invention, a vaccine adjuvant for promoting a T lymphocyte-specific immune response includes a polynucleotide encoding an IL-12 protein and a polynucleotide encoding an IL-21 protein as active ingredients.
In particular, the vaccine adjuvant may include at least one selected from the group consisting of the followings:

- one to three vectors, each of which include: polynucleotides that respectively encode a p35 chain (IL-12p35) and a p40 chain (IL-12p40) that constitute the IL-12 protein; and a polynucleotide that encodes the IL-21 protein; and
- a mRNA molecules, each of which encode the IL-12p35, IL-12p40, and IL-21 proteins.

Furthermore, it is possible that in the above-described vaccine adjuvant, part of the IL-21p35, IL-12p40, and IL-21 may be included as proteins and the rest may be used as a mixture of heterogeneous molecules, such as expression vector and/or mRNA molecules.
In particular, the one to three vectors may include a gene construct in which the polynucleotide is operably linked to a control sequence (e.g., a promoter) so that the IL-21p35, IL-12p40, and IL-21 can be expressed. The vaccine adjuvant may be constituted as one to three vectors such that polynucleotides, each of which encode the IL-21p35, IL-12p40, and IL-21, are inserted into an individual expression vector (a triple vector system), or are inserted into one or two expression vectors (a single vector or dual vector system). A specific exemplary embodiment of such a single vector system to a triple vector system is as follows:

- i) a 4^thexpression vector, which includes a 1^stgene construct to a 3^rdgene construct in which polynucleotides that encode the IL-21p35, IL-12p40, and IL-21 proteins, respectively, are each operably linked to a promoter;
- ii) a 2^ndexpression vector to a 4^thexpression vector, which include the 1^stgene construct to the 3^rdgene construct, respectively;
- iii) a 5^thexpression vector and a 6^thexpression vector, each of which include two of the 1^stgene construct to the 3^rdgene construct and the remaining one gene construct, respectively;
- iv) a fusion protein in which an IL-21 is linked to any one of the IL-12p35 and the IL-12p40; and a peptide between the IL-12p35 and the IL-12p40 which is not included in the fusion protein;
- v) a 7^thexpression vector, which includes a 4^thgene construct in which a polynucleotide encoding the fusion protein of iv) is operably linked to a promoter, and a 5^thgene construct, in which a polynucleotide encoding the peptide of iv) is operably linked to a promoter;
- vi) an 8^thexpression vector and a 9^thexpression vector, which include the 4^thgene construct and the 5^thgene construct, respectively;
- vii) a 10^thexpression vector which includes: a 6^thgene construct in which at least two of the polynucleotides that encode the IL-12p35, IL-12p40, and IL-21 proteins, respectively, are linked to an internal ribosome entry site (IRES); and optionally a 7^thgene construct in which the remaining polynucleotide, among the three polynucleotides, that is not included in the 6^thgene construct is operably linked to a promoter; and
- viii) an 11^thexpression vector that includes the 9^thgene construct; and a 12^thexpression vector, which optionally includes the 7^thgene construct.

The vaccine adjuvant may promotes a T lymphocyte-specific immune response without inducing a B lymphocyte-specific immune response.
In the vaccine adjuvant, the IL-12p35 protein may consist of an amino acid sequence which has at least 90%, and preferably at least 95% of a sequence homology with the human IL-12p35 that consists of an amino acid sequence of SEQ ID NO: 1, and it is also possible to use the IL-12p35 derived from non-humans (e.g., primates or simians) that have a high level of homology not to induce any immune response in the human body. The IL-12p40 protein may consist of an amino acid sequence which has at least 90%, and preferably at least 95% of a sequence homology with the human IL-12p40 that consists of an amino acid sequence of SEQ ID NO: 2, and it is also possible to use the IL-12p40 derived from non-humans (e.g., primates or simians) that have a high level of homology not to induce any immune response in the human body. It is also possible to use the IL-12p35 and the IL-12p40 as sequences described in KR Patent No. 0399728. The IL-21 protein may consist of an amino acid sequence which has at least 90%, and preferably at least 95% of a sequence homology with the human IL-21 that consists of an amino acid sequence of SEQ ID NO: 3, and it is also possible to use the IL-21 derived from non-humans (e.g., primates or simians) that have a high level of homology not to induce any immune response in the human body.
The vaccine adjuvant may further include at least one selected from the group consisting of:

- an MIP-1α gene construct, in which a polynucleotide encoding the MIP-1α protein is operably linked to a promoter;
- a complex gene construct, in which a polynucleotide encoding the MIP-1α protein is operably linked to at least one among the polynucleotides encoding each of the IL-12p35, IL-12p40, and IL-21 proteins, by a polynucleotide encoding an IRES or linker peptide; and
- an mRNA molecule encoding the MIP-1α protein.

In the vaccine adjuvant, the polynucleotide encoding the MIP-1α protein may be operably linked to polynucleotides encoding each of the IL-12p35, IL-12p40, and IL-21 proteins via a polynucleotide or IRES that encodes a peptide linker, or may be provided in the form of a separate gene construct. That is, the MIP-1α gene construct may be included in a separate expression vector, or the MIP-1α gene construct may be included in any one or more vector among the one to three vectors, each of which include: polynucleotides that respectively encode a p35 chain (IL-12p35) and a p40 chain (IL-12p40) that constitute the IL-12 protein; and a polynucleotide that encodes the IL-21 protein. That is, the MIP-1α gene construct may be included in at least one expression vector selected from the group consisting of a 1^thexpression vector to a 12^thexpression vector described in the exemplary embodiment with regard to the vaccine adjuvant.
In the vaccine adjuvant, the MIP-1α protein may consist of an amino acid sequence which has at least 90%, and preferably at least 95% of a sequence homology with the MIP-1α protein that consists of an amino acid sequence of SEQ ID NO: 10, and it is also possible to use the MIP-1α protein derived from non-humans (e.g., primates or simians) that have a high level of homology not to induce any immune response in the human body.
In another aspect of the present invention, there is provided a vaccine adjuvant including an IL-12 protein and an IL-21 protein as active ingredients.
In the vaccine adjuvant, the IL-12 protein may consist of a p35 chain (IL-12p35) and a p40 chain (IL-12p40).
The vaccine adjuvant may further include MIP-1α protein.
The IL-12 protein, IL-21 protein, and MIP-1α are as described above.
As used herein, the term “operably linked to” means that the nucleic acid sequence of interest (e.g., in vitro transcription/translation system or in a host cell) is linked to the control sequence such that the heterologous nucleic acid sequence can be expressed.
The term “control sequence” is a term that includes a promoter, an enhancer, and other control sequences (e.g., a polyadenylated signal). The control sequence includes: one which directs such that the heterologous nucleic acid is constitutively expressed in many host cells, one which directs such that the heterologous nucleic acid is expressed only in cells of a specific tissue (e.g., a tissue-specific control sequence), and one which directs such that expression is induced by a particular signal (e.g., an inducible control sequence). One of ordinary skill in the art will be able to understand that the design of the expression vector may depend on factors, such as the choice of a host cell to be transformed, the level of protein expression desired, etc. The expression vector of the present invention may be introduced into a host cell to express the fusion protein. Control sequences that allow expression in the eukaryotic and prokaryotic cells are well known to those skilled in the art. As described above, these control sequences include those which are normally in charge of the initiation of transcription and a poly-A signal which is in charge of selective termination of transcription of a transcript and stabilization thereof. Additional control sequences may include translation enhancing factors and/or native-combined or heterologous promoter regions, in addition to transcription control factors. For example, possible control sequences that enable expression in a mammalian host cell may include CMV-HSV thymidine kinase promoter, SV40, RSV-promoter (Rous sarcoma virus), human elongation factor 1α-promoter, glucocorticoid-inducible MMTV-promoter (Moloney mouse tumor virus), metallothionein-inducible or tetracycline-inducible promoter, or an amplifying agent such as a CMV amplifying agent or an SV40-amplifying agent. For expression in neurons, the use of neurofilament-promoter, PGDF-promoter, NSE-promoter, PrP-promoter, or thy-1-promoter is being considered. The promoters are well known in the art and are described in the literature (Charron, J Biol. Chem. 270: 25739-25745). For expression in prokaryotic cells, many promoters including lac-promoter, tac-promoter, and trp promoter are disclosed. These control sequences may also include a transcription termination signal (e.g., SV40-poly-A region or TK-poly-A region) in the downstream of the polynucleotide in accordance with an exemplary embodiment of the present disclosure, in addition to those factors which can initiate transcription. In the present disclosure, suitable expression vectors are well known in the art, for example, Okayama-Berg cDNA expression vector pcDV1 (Parmacia), pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), pSPORT1 (GIBCO BRL), pGX27 (KR Patent No. 1442254), pX(Pagano (1992) Science 255, 1144-1147), yeast two-hybrid vector, for example, pEG202 and dpJG4-5 (Gyuris (1995) Cell 75, 791-803) or eukaryotic expression vector, for example, lambda gt11 or pGEX (Amersham-Pharmacia). The vector may further include a polynucleotide encoding a secretion signal peptide, in addition to the nucleic acid molecules of the present disclosure. The secretion signal peptides are well known to those skilled in the art. Additionally, according to the expression system used, a leader sequence which can direct the fusion protein to the cell compartment is combined with the coding sequence of the polynucleotide according to an exemplary embodiment of the present disclosure, and preferably a leader sequence which can directly secrete the translated protein or a protein thereof into the pericytoplasmic or extracellular medium.
In addition, vectors of the present invention can be prepared, for example, by standard recombinant DNA techniques. Examples of the standard recombinant DNA techniques include ligation of blunt ends and sticky ends, restriction enzyme treatment to provide appropriate ends, removal of phosphate groups by alkaline phosphatase treatment to prevent inappropriate bindings, enzymatic linkage by T4 DNA ligase, etc. The vector of the present invention may be prepared by recombination of the DNA encoding the signal peptide obtained by chemical synthesis or genetic recombination technology or the DNA encoding the IL-12 and IL-21 proteins of the present invention to the vector which include an appropriate control sequence. The vector containing the control sequence may be purchased or manufactured commercially, and in an exemplary embodiment of the present disclosure, pGX27, which is a vector for the preparation of DNA vaccines, was used.
The expression vector according to an exemplary embodiment of the present invention may be an expression vector that can express the fusion protein, and the expression vector may represent without limitation any form of a plasmid vector, a viral vector, a cosmid vector, a phagemid vector, an artificial human chromosome, etc.
As used herein, the term “fusion protein” refers to a recombinant protein in which two or more proteins or domains in charge of specific functions in the protein are linked so that each protein or domain can be in charge of the intrinsic function. Conventionally, a linker peptide with a flexible structure may be inserted between the two or more proteins or domains. The linker peptide may include (G4S)_n(unit: SEQ ID NO: 50, n is an integer of 1 to 10), (GS)_n(n is an integer of 1 to 10), (GSSGGS)_n(unit: SEQ ID NO: 51, n is an integer of 1 to 10), KESGSVSSEQLAQFRSLD (SEQ ID NO: 52), EGKSSGSGSESKST (SEQ ID NO: 53), GSAGSAAGSGEF (SEQ ID NO: 54), (EAAAK)_n(unit: SEQ ID NO: 55, n is an integer of 1 to 10), CRRRRRREAEAC(SEQ ID NO: 56), A(EAAAK)₄ALEA(EAAAK)₄A (SEQ ID NO: 57), GGGGGGGG (SEQ ID NO: 58), GGGGGG (SEQ ID NO: 59), AEAAAKEAAAAKA (SEQ ID NO: 60), PAPAP (SEQ ID NO: 61), (Ala-Pro). (n is an integer of 1 to 10), VSQTSKLTRAETVFPDV (SEQ ID NO: 62), PLGLWA (SEQ ID NO: 63), TRHRQPRGWE (SEQ ID NO: 64), AGNRVRRSVG (SEQ ID NO: 65), RRRRRRRR (SEQ ID NO: 66), GFLG (SEQ ID NO: 67), GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 68), etc.
The expression vector may further include a polynucleotide that encodes one or two or more of peptides for enhancing immunity, and the peptides for enhancing immunity may be CD28, inducible costimulator (ICOS), cytotoxic T lymphocyte associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), B and T lymphocyte associated protein (BTLA), death receptor 3 (DR3), 4-1BB, CD2, CD40, CD30, CD27, signaling lymphocyte activation molecule (SLAM), 2B4 (CD244), natural-killer group 2, member D (NKG2D)/DNAX-activating protein 12 (DAP12), T-Cell immunoglobulin and mucin domain containing protein 1 (TIM1), TIM2, TIM3, TIGIT, CD226, CD160, lymphocyte activation gene 3 (LAG3), B7-1, B7-H1, glucocorticoid-induced TNFR family related protein (GITR), fins-like tyrosine kinase 3 ligand (Flt3 ligand), flagellin, herpesvirus entry mediator (HVEM), or a cytoplasmic domain of OX40L[ligand for CD134(OX40) and CD252], or a linker of at least two thereof.
The expression vector may further include a polynucleotide encoding a secretory signal sequence, and the secretory signal sequence induces the secretion of recombinant proteins expressed in cells outside the cell, and may be tissue plasminogen activator (tPA) signal sequence, herpes simplex virus glycoproteins (HSV gDs) signal sequence, or a growth hormone signal sequence.
In another aspect of the present invention, there is provided a vaccine composition for prevention and treatment of virus infection includes, as active ingredients, the vaccine adjuvant and an antigen derived from infectious virus, an antigen gene construct in which the polynucleotide encoding the antigen is operably linked to a promoter, or an expression vector including the gene construct.
In the vaccine composition, the antigen derived from infectious virus may be an antigen derived from Epstein-Barr virus (EBV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), Hantaan virus, cytomegalovirus (CMV), human immunodeficiency virus (HIV), influenza virus, human papillomavirus (HPV), poliovirus, ebola virus, rotavirus, dengue virus, West Nile virus, yellow fever virus, adenovirus, Japanese encephalitis virus, BK virus, smallpox virus, Zika virus, severe fever with thrombocytopenia syndrome (SFTS) virus, or herpes simplex virus (HSV).
According to still another aspect of the present disclosure, there is provided a vaccine composition for cancer treatment includes, as active ingredients, the vaccine adjuvant and a cancer antigen, a polynucleotide encoding the cancer antigen, an antigen gene construct in which the polynucleotide is operably linked to a promoter, or an expression vector including the gene construct.
In the vaccine composition for cancer treatment, the cancer antigen may be a human papillomavirus (HPV)-derived antigen, a carcinoembryonic antigen, a prostate-specific membrane antigen (PSMA), Her2/neu, MUC-1, BCR/ABL, α-fetoprotein (AFP), an Epstein-Barr virus (EBV)-derived antigen, a human hepatitis B virus (HBV)-derived antigen, a human hepatitis C virus (HCV)-derived antigen, cancer antigen-125 (CA-125), cancer antigen-72-4 (CA-72-4), cancer antigen-15-3 (CA-15-3), or cancer antigen-19-9 (CA-19-9).
The vaccine composition may further include a pharmaceutically acceptable adjuvant, an excipient, or a diluent, in addition to the carrier.
As used herein, the term “pharmaceutically acceptable” refers to a composition which is physiologically acceptable and does not normally cause an allergic reaction (e.g., a gastrointestinal disorder, dizziness, etc.) or a similar reaction thereof when administered to humans. Examples of the carrier, excipient, and diluent may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, fillers, anti-coagulants, lubricants, humectants, fragrances, emulsifiers, preservatives, etc. may be further included.
The vaccine composition may further include a conventionally used vaccine adjuvant, in addition to the vaccine adjuvant above. As the conventionally used vaccine adjuvant, aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), MF59, virosome, ASO4 [a mixture of aluminum hydroxide and monophosphoryl lipid A (MPL)], ASO3 (a mixture of DL-α-tocopherol, squalene, and polysorbate 80 (an emulsifier)), CpG, Flagellin, Poly I:C, ASO1, AS02, ISCOMs, ISCOMMATRIX, etc. may be used.
As used herein, the term “adjuvant” or “vaccine adjuvant” refers to a pharmaceutical or immunological preparation being administered for the purpose of improving the immune response of a vaccine.
Additionally, the vaccine adjuvant or the vaccine composition containing the vaccine adjuvant according to an exemplary embodiment of the present invention may be formulated using methods known in the art so as to enable rapid release, or sustained or delayed release of the active ingredient when administered to a mammal. These preparations may include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powders.
The vaccine adjuvant or the vaccine composition containing the vaccine adjuvant according to an exemplary embodiment of the present invention may be administered in various routes including, for example, oral, parenteral (e.g., suppository, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, nasal, spinal canals, or may be administered using an implantable device for sustained, continuous, or repeated release. The number of administrations may be administered once or several times a day within a desired range, and the administration period is not particularly limited.
The vaccine adjuvant or the vaccine composition containing the vaccine adjuvant according to an exemplary embodiment of the present invention may be administered by conventional systemic or topical administration (e.g., intramuscular injection or intravenous injection), if provided as a DNA vaccine composition, the vaccine composition may be most preferably injected using an electroporator. As the electroporator, electroporators for injecting a commercially available DNA drug into the body (e.g., Glinporator™ of IGEA, Italy, CUY21EDIT of JCBIO, Korea, SP-4a of Supertech, Switzerland, OrbiJector of SLVAXiGEN, Korea, etc.) may be used.
The administration route of the vaccine adjuvant or the vaccine composition containing the vaccine adjuvant according to an exemplary embodiment of the present invention may be administered via any conventional route as long as the vaccine composition can reach the target tissue. The administration route may include, but is not limited to, parenteral administration (e.g., intraperitoneal, intravenous, intramuscular, subcutaneous, and intrathecal administration).
The administration route of the vaccine adjuvant or the vaccine composition containing the vaccine adjuvant according to an exemplary embodiment of the present invention may be formulated in a suitable form together with a commonly available pharmaceutically acceptable carrier. The pharmaceutically acceptable may include, for example, water, suitable oils, saline, parenteral vehicles (e.g., aqueous glucose, glycols, etc.) and may further contain stabilizers and preservatives. Suitable stabilizers may include antioxidants (e.g., sodium hydrogen sulfite, sodium sulfite, or ascorbic acid). Suitable preservatives may include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Additionally, the composition according to the present invention may appropriately include a suspending agent, a dissolution adjuvant, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjusting agent, an analgesic agent, a buffering agent, an antioxidant, etc., depending on the administration method and preparation, if necessary. Suitable examples of suitable pharmaceutically acceptable carriers and preparations suitable for the present disclosure, including those exemplified above, are described in detail in the literature [Remington's Pharmaceutical Sciences, New Edition].
The administration dose of the vaccine composition for a patient may vary depending on many factors including the patient's height, body surface area, age, the particular compound being administered, sex, time and route of administration, general health conditions, and other drugs being administered concurrently. The pharmaceutically active DNA may be administered in an amount of 100 ng/body weight (kg) to 10 mg/body weight (kg), more preferably from 1 μg/kg to 500 μg/kg (body weight), and most preferably from 5 μg/kg to 50 μg/kg (body weight), and the administration dose may be adjusted considering the factors.
In addition, the vaccine composition of the present invention may be administered in a pharmaceutically effective amount.
As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient for the treatment of diseases at a reasonable benefit/risk ratio applicable to medical treatment, and the level of the effective dose may be determined based on the factors including the kind of subject, severity of illness, age, sex, drug activity, drug sensitivity, administration time, administration route and dissolution rate, length of treatment, factors including drug(s) to be used simultaneously in combination, and other factors well known in the medical field. The vaccine composition of the present invention may be administered at a dose of 0.1 mg/kg to 1 g/kg, and more preferably from 1 mg/kg to 500 mg/kg. Meanwhile, the administration dose may be appropriately adjusted according to age, sex, and health conditions of the patient.
In addition, according to still another aspect of the present disclosure, there is provided a method for stimulating a T lymphocyte-specific immune response by a vaccine composition including administering, to an individual, the vaccine adjuvant along with a vaccine composition, or before or after the administration of the vaccine composition.
In the method, the vaccine adjuvant may be administered by in vivo electroporation.
The method is characterized in that the method selectively induces only a T lymphocyte-specific immune response without inducing a B cell-specific immune response of the individual.
Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, the present invention is not limited to the Examples and Experimental Examples described below, but may be implemented in various other forms. The following Examples and Experimental Examples are provided to enable the disclosure of the present invention to be complete and to fully convey the scope of the invention to those skilled in the art to which the present invention belongs.

Example 1: Preparation of Expression Vectors for Human IL-12 and IL-21

1-1: Single Vector System
The present inventors have designed a single vector system such that IL-12 and IL-21 are expressed via a single vector.
For this purpose, specifically, the present inventors have prepared a gene construct by linking the polynucleotides (SEQ ID NOS: 4 and 5) which encode each of two subunits of human IL-12 protein (i.e., the hIL-12p35 polypeptide consisting of an amino acid sequence of SEQ ID NO: 1 and the hIL-12p40 polypeptide consisting of an amino acid sequence of SEQ ID NO: 2) to an EMCV-derived internal ribosome entry site (IRES) having a nucleic acid sequence of SEQ ID NO: 6; and sequentially linking the RSV promoter (pRSV) of SEQ ID NO: 7 and a polynucleotide (SEQ ID NO: 8), which encodes human IL-21 protein (hIL-21) consisting of an amino acid sequence of SEQ ID NO: 3, to the 3′-end of a polynucleotide encoding the hIL-12p40 polypeptide; and then inserting the gene construct into a multiple cloning site of the pGX-27 vector (KR Patent No. 1442254), thereby preparing a vector according to an exemplary embodiment of the present disclosure. The thus prepared vector was named as “hBD-121” (FIG. Ta).
1-2: Dual Vector System
The present inventors designed a dual vector system such that IL-12 and IL-21 are inserted into separate vectors to be expressed.
The dual vector system is prepared as follows:
A polynucleotide (SEQ ID NO: 4) encoding the hIL-12p35 polypeptide and a polynucleotide (SEQ ID NO: 5) encoding the hIL-12p40 polypeptide are linked to an EMCV-IRES having a nucleic acid sequence of SEQ ID NO: 6; and the resultant is inserted into a multiple cloning site of the pGX-27 vector; and likewise, a polynucleotide (SEQ ID NO: 8) encoding the human IL-21 protein (hIL-21) is also inserted into a multiple cloning site of the pGX-27 vector; and thereby a dual vector system is prepared.
1-3: Triple Vector System
Since the IL-12 is a dimer protein consisting of a hIL-12p35 polypeptide and a hIL-12p40 polypeptide, the hIL-12p35 polypeptide and the hIL-12p40 polypeptide may be expressed from an independent vector. As such, according to an exemplary embodiment of the present disclosure, the hIL-12p35 polypeptide, hIL-12p40 polypeptide, and IL-21 may be expressed via each of three independently constituted vectors, and for convenience purposes, this was named as “triple vector system”.
The triple vector system may be prepared as follows:
The polynucleotides (SEQ ID NOS: 4, 5, and 8) encoding each of the hIL-12p35 polypeptide, hIL-12p40 polypeptide, and hIL-21 are inserted into a multiple cloning site of the pGX-27 vector, and thereby preparing a triple vector system.

Example 2: Preparation of Expression Vectors for Human IL-12, IL-21, and MIP-1α

A gene construct was prepared by linking the polynucleotides (SEQ ID NOS: 4 and 5), which encode each of the hIL-12p35 polypeptide consisting of an amino acid sequence of SEQ ID NO: 1 and the hIL-12p40 polypeptide consisting of an amino acid sequence of SEQ ID NO: 2, to an EMCV-derived internal ribosome entry site (IRES) having a nucleic acid sequence of SEQ ID NO: 6; and sequentially linking human EF-1α promoter (pEF-1α), which consists of a nucleic acid sequence of SEQ ID NO: 9, and a polynucleotide (SEQ ID NO: 11), which encodes human MIP-1α protein (hMIP-1α) consisting of an amino acid sequence of SEQ ID NO: 10, to a polynucleotide, to which the RSV promoter (pRSV) of SEQ ID NO: 7 and a polynucleotide (SEQ ID NO: 8), which encodes human IL-21 protein (hIL-21) consisting of an amino acid sequence of SEQ ID NO: 3, are sequentially linked; and inserting the gene construct into a multiple cloning site of the pGX-27 vector; and the thus prepared vector was named as “hBD-121A” (FIG. 1 b ).

Example 3: Preparation of Expression Vectors for Mouse IL-12 and IL-21

A gene construct was prepared by linking the polynucleotides (SEQ ID NOS: 14 and 15) which encode each of two subunits of mouse IL-12 protein (i.e., the mIL-12p35 polypeptide consisting of an amino acid sequence of SEQ ID NO: 12 and the mIL-12p40 polypeptide consisting of an amino acid sequence of SEQ ID NO: 13) to an EMCV-derived internal ribosome entry site (IRES) having a nucleic acid sequence of SEQ ID NO: 6; and sequentially linking the RSV promoter (pRSV) of SEQ ID NO: 7 and a polynucleotide (SEQ ID NO: 17), which encodes mouse IL-21 protein (mIL-21) consisting of an amino acid sequence of SEQ ID NO: 16, to the 3′-end of a polynucleotide encoding the mIL-12p40 polypeptide; and inserting the gene construct into a multiple cloning site of the pGX-27 vector, thereby preparing a vector according to an exemplary embodiment of the present disclosure. The thus prepared vector was named as “mBD-121” (FIG. 1 a ).

Example 4: Preparation of Expression Vectors for Mouse IL-12, IL-21 and MIP-1α

A gene construct was prepared by linking the polynucleotides (SEQ ID NOS: 14 and 15), which encode each of the mIL-12p35 polypeptide consisting of an amino acid sequence of SEQ ID NO: 12 and the mIL-12p40 polypeptide consisting of an amino acid sequence of SEQ ID NO: 13, to an EMCV-derived internal ribosome entry site (IRES) having a nucleic acid sequence of SEQ ID NO: 6; and sequentially linking human EF-1α promoter (pEF-1α), which consists of a nucleic acid sequence of SEQ ID NO: 9, and a polynucleotide (SEQ ID NO: 19), which encodes mouse MIP-1α protein (mMIP-1α) consisting of an amino acid sequence of SEQ ID NO: 18, to the 3′-end of a polynucleotide encoding the mIL-12p40 polypeptide (SEQ ID NO: 815), to which the RSV promoter (pRSV) of SEQ ID NO: 7 and a polynucleotide (SEQ ID NO: 17), which encodes mouse IL-21 protein (mIL-21) consisting of an amino acid sequence of SEQ ID NO: 16, are sequentially linked; and inserting the gene construct into a multiple cloning site of the pGX-27 vector; and the thus prepared vector was named as “mBD-121A” (FIG. 1 b ).

Example 5: Preparation of HPV DNA Vaccine Construct

To prepare a 14-valent HPV DNA vaccine, the present inventors have obtained polynucleotides encoding the N-terminal fragment and the C-terminal fragment of E6 and E7 antigens of each type obtained by PCR, in order to express the E6 and E7 antigens in the form of a shuffled fusion protein, in which the early expression proteins E6 and E7 of HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 belong to a high-risk group, and then have linked the polynucleotides by linking in the order disclosed in FIG. 2 and Table 1, thereby preparing three gene constructs. Each of the three gene constructs was inserted into a pGX-27 vector, thereby preparing a HPV DNA construct. Each of the three gene constructs was named as BD-14A, BD-14B, and BD-14C, respectively, and the composition containing these three vectors was named as BD-14. The reason why the polyvalent HPV DNA vaccine construct was prepared as a triple vector system was because the pGX-27 vector is inefficient in capacity when the size of the gene construct to be inserted is too large.
As shown in FIG. 2 and Table 1, the E6 and E7 antigens of each HPV type were constructed in such a structure where these antigens are divided into the N-terminal fragment and the C-terminal fragment in which a partial sequence is overlapping, respectively, and then a fusion polypeptide, where the C-terminal fragment of E7 is linked to the end of the N-terminal fragment of E6, and a fusion polypeptide, where the C-terminal fragment of E6 is linked to the end of the N-terminal fragment of E7, are linked again by a (GS)₅linker peptide, so that 4 to 5 antigen units of each type are linked to the (GS)₅linker to be included in a single vector. The kinds of each subtype to be inserted into one expression vector are exemplarily shown in Table 1, but they are for illustrative purposes only and may be prepared in any other order.

TABLE 1

Constitution of 14-valent HPV DNA vaccine construct of the present disclosure

SEQ ID NO

Constituting				Nucleic
Element		Specific Structure	Protein	Acid	Origin

Common	linker	(GS)₅	20	21	N/A
Element	tPA	tPA_1-22	22	23	Uniprot: P00750
	Flt3L	Flt3L_{27-182, Δ1-26}	24	25	Uniprot: P49771
BD-14A	16	16E6N_1-85-16E7C_41-105-(GS)₅-	26	27	GenBank:
	E6E7	16E7N_1-60-16E6C_66-158			K02718.1
	18	18E6N_1-85-18E7C_41-98-(GS)₅-			GenBank:
	E6E7	18E7N_1-60-18E6C_66-158			X05015.1
	35	35E6N_1-78-35E7C_42-99-(GS)₅-			GenBank:
	E6E7	35E7N_1-61-35E6C_59-149			X74477.1
	45	45E6N_1-85-45E7C_41-106-(GS)₅-			GenBank:
	E6E7	45E7N_1-60-45E6C_66-158			X74479.1
	58	58E6N_1-85-58E7C_41-98-(GS)₅-			GenBank:
	E6E7	58E7N_1-60-58E6C_66-149			D90400.1
BD-14B	31	31E6N_1-85-31E7C_42-98-(GS)₅-	28	29	GenBank:
	E6E7	31E7N_1-61-31E6C_66-149			J04353.1
	33	33E6N_1-85-33E7C_42-96-(GS)₅-			GenBank:
	E6E7	33E7N_1-61-33E6C_66-149			M12732.1
	06	6E6N_1-85-6E7C_42-98-(GS)₅-			GenBank:
	E6E7	6E7N_1-61-6E6C_66-150			X00203.1
	11	11E6N_1-85-11E7C_42-98-(GS)₅-			GenBank:
	E6E7	11E7N_1-61-11E6C_66-150			M14119.1
	52	52E6N_1-85-52E7C_41-99-(GS)₅-			GenBank:
	E6E7	52E7N_1-60-52E6C_66-148			X74481.1
BD-14C	39	39E6N_1-85-39E7C_44-109-(GS)₅-	30	31	GenBank:
	E6E7	39E7N_1-63-51E6C_66-158			M62849.1
	51	51E6N_1-83-51E7C_45-101-(GS)₅-			Uniprot:
	E6E7	51E7N_1-64-51E6C_64-151			P26554(E6),
					P26558(E7)
	56	56E6N_1-86-56E7C_48-105-(GS)₅-			Uniprot:
	E6E7	56E7N_1-67-56E6C_67-155			P24836(E6),
					P36833(E7)
	59	59E6N_1-85-59E7C_50-107-(GS)₅-			GenBank:
	E6E7	59E7N_1-69-59E6C_66-160			CAA54849.1(E6),
					CAA54850.1(E7)

Example 6: Preparation of CMV DNA Vaccine Construct

To prepare a CMV DNA vaccine construct, the present inventors prepared a polynucleotide encoding the proteins in which the CMV glycoprotein B (gB) and the pp65 protein are included, and then inserted the polynucleotide into a multiple cloning site of the pGX-27 vector, and thereby preparing a CMV DNA vaccine construct (FIG. 3 ).

Example 7: Preparation of HBV DNA Vaccine Construct

To prepare a HBV DNA vaccine construct, the present inventors prepared a HBV DNA vaccine construct so as to express preS1, preS2, S antigen, a surface antigen, and a nuclear antigen, and then inserted the HBV DNA vaccine construct into the pGX-27 vector.

Example 8: Preparation of HSV-2 DNA Vaccine Construct

To prepare a HSV-2 DNA vaccine construct, the present inventors divided the UL39 antigen of HSV-2 into 5 fragments (i.e, N1: UL39_{21-154(Δ78-104)}, C2: UL39_1117-1142, N2: UL39_165-227, N4-C1: UL39_398-1116, and N3: UL39_208-398), and then inserted a shuffled UL-39 antigen of HSV-2 (arrayed by shuffling the 5 fragments) and a gene construct encoding the shuffled UL-39 into a multiple cloning site of the pGX-27 vector, and thereby preparing a HSV-2 DNA vaccine construct.
Specifically, the shuffled-UL39 plasmid DNA was prepared by inserting, into the pGX-27 plasmid vector, a gene construct which includes a polynucleotide (SEQ ID NO: 38) that encodes the form sequentially connected in the order of UL39-N1, UL39-C2, UL39-N2, UL39-N4-C1, and UL39-N3, based on the form divided into UL39-N1 (SEQ ID NO: 32), UL39-C2 (SEQ ID NO: 33), UL39-N2 (SEQ ID NO: 34), UL39-N4-C1 (SEQ ID NO: 35), and UL39-N3 (SEQ ID NO: 36) (FIG. 4 a ), and the shuffled-UL39 plasmid DNA was named as “BD-02B”.
Furthermore, the present inventors prepared a tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA by inserting a gene construct into the pGX-27 vector, in which the gene construct includes a polynucleotide (SEQ ID NO: 43) encoding the tPA-Flt3L-gD-ICP0-ICP4 fusion protein (SEQ ID NO: 42), in which a HSV-2 antigen other than the shuffled UL39 antigen, that is, a glycoprotein D (gD_26-349) in which a signal sequence (gD_1-25) and a transmembrane domain (gD_350-363) are removed (SEQ ID NO: 39), an infected cell polypeptide 0 (ICP0_Δ510-516, SEQ ID NO: 40) in which a nuclear localization sequence (NLS, ICP0_510-516) is removed, and an infected cell polypeptide 4 (ICP4_Δ767-1318, SEQ ID NO: 41) in which the RS1.3 part (ICP_767-1318) is removed are connected (FIG. 4 b ). Both the above two plasmid DNAs are plasmid DNAs which are designed to be expressed in the form of a fusion protein, in which a tPA secretory signal sequence (SEQ ID NO: 22) which is codon optimized for efficient expression, and an FMS-like tyrosine kinase 3 ligand (Flt3L, SEQ ID NO: 24) (i.e., an immunoactive protein) are added to the N-terminus. The thus prepared plasmid DNA for a DNA vaccine was named as “BD-02C” and the DNA vaccine composition consisting of the “BD-02B” and the “BD-02C” were named as “BD-02”.

Example 9: Preparation of SFTS DNA Vaccine Construct

To express glycoproteins N and C (GnGc, SEQ ID NO: 44), nucleocapsid (NP, SEQ ID NO: 45), and nonstructural (NS) protein (SEQ ID NO: 46) antigens except the antigen RNA dependent RNA polymerase (RdRP) of SFTS, the present inventors amplified the polynucleotides encoding each of the proteins (SEQ ID NOS: 47 to 49) by PCR using the SFTS virus genome as a template. Each of the amplified polynucleotides was operably linked to CMV promoter (pCMV), RSV promoter (pRSV), and EF-1α promoter (pEF-1α), and thereby preparing a recombinant plasmid DNA (SFTS plasmid DNA), which is an SFTS DNA vaccine (FIG. 5 ).

Experimental Example 1: Confirmation of Potentials of IL-12 and IL-21 as Vaccine Adjuvant

The present inventors performed a preliminary experiment to confirm the potentials of IL-12 and IL-21 as the vaccine adjuvant in the form of a protein and DNA. For this purpose, specifically, the immune responses were examined by administering, to an individual, a HPV 2-valent DNA vaccine alone and in combination with IL-12 and/or IL-21 to HPV types 16 and 18.
Specifically, 2 μg each of type 16/18 HPV 2-valent DNA vaccine construct described in KR Patent Laid-open Publication No. 10-2017-0045254 was administered to the femoral muscles of C57BL/6 mice twice at two-week intervals using OrbiJector® (SLVAXiGEN, Korea) by in vivo electroporation, in combination with 2 μg each of a mIL-12 construct and/or a mIL-21 construct. Then, two weeks after the 2^ndadministration day, spleens were extracted and the number of spleen immunocytes reactive to each type of HPV 16 and HPV18 was counted (FIG. 6 ).
As a result, as shown in FIG. 6 , it was confirmed that the cells exhibiting an immune response in mice simultaneously treated with IL-12 and IL-21 were significantly increased, compared to the treatment with IL-12 or IL-21 alone, and in particular, it was confirmed that the effect was remarkable in type 18 HPV.
These results suggest that IL-12 and IL-21 are cytokines that can synergistically increase the immune response of the vaccine.

Experimental Example 2: Analysis of BD-121 Expression

2-1: ELISA Assay
The present inventors transformed each of the hBD-121 construct and the mBD-121 construct prepared in Examples 2 and 4 according to an exemplary embodiment of the present disclosure, respectively, into cells and examined whether IL-12 and IL-21 were normally expressed in the transformed cells. Specifically, the COS-7 cell line was inoculated on each 100 mm culture dish and cultured for 16 hours, transformed with each of an empty vector (mock plasmid DNA), the hBD-121 plasmid DNA prepared in Example 2-1, and the mBD-121 plasmid DNA prepared in Example 4, using Lipofectamine 2000. Each transformant was cultured in an incubator (37° C., CO₂) for 3 days, and the culture supernatant of the COS-7 cells under each condition was recovered and used as a sample. The IL-12 and IL-21 proteins present in each sample were quantified by ELISA assay using the antibodies (IL-12: R&D Systems, Cat #D1200, IL-21: BioLegend, Cat #433808) which specifically recognize IL-12 and IL-21, respectively (FIGS. 7 a and 7 b ).
As a result, as shown in FIG. 7 a , when hIL-12 was introduced in an amount of 4 μg of DNA, the expression level of the proteins in the sample where the hBD-121 plasmid DNA was introduced was greater than 4,000 pg/mL thus confirming that these proteins were normally expressed, whereas when hIL-21 was introduced in an amount of 4 μg of DNA, the expression level of the proteins in the sample was as high as being close to 200 ng/mL thus confirming these proteins were expressed at high level. Furthermore, as shown in FIG. 7 b , the mouse construct showed a result similar to that of the human construct. Meanwhile, in the control group where the empty vector was introduced, both proteins were not expressed at all thus confirming that the vaccine adjuvant expression system of the present invention acted normally.
1-2: Western Blot Analysis
The present inventors performed SDS-PAGE electrophoresis using the cell lysate of the cells obtained in Experimental Example 1-1, transferred onto a nylon membrane, and performed western blot analysis using an anti-IL-12A antibody (Abcam, Cat #ab131039), an anti-IL-12B antibody (Abcam, Cat #ab133752), and an anti-IL-21 antibody (Abcam, Cat #ab5978) (FIG. 7 c ).
As a result, as shown in FIG. 7 c , it was confirmed that both IL-12 and IL-21 were normally expressed by the transfection of BD-121 plasmid DNA according to an exemplary embodiment of the present disclosure.

Experimental Example 3: Analysis of Effect of BD-121 on Improving Immune Response Against Various Viruses

Subsequently, from the results of Experimental Example 2, the present inventors examined whether a BD-121 construct according to an exemplary embodiment of the present invention can actually improve immune responses against various viruses.
3-1: T Cell-Specific Immune Response and Antibody Response Against CMV
First, the present inventors inoculated once the CMV DNA vaccine construct (0.5 μg), which was prepared in Example 6, alone or in combination with BD-121 (0.5 μg), to the femoral muscles of C57BL/6 mice using OrbiJector® (SLVAXiGEN, Korea) by in vivo electroporation. In particular, as comparative groups, expression vectors which express IL-12 and IL-21 (i.e., constituting elements of BD121), respectively, were administered alone or in combination (IL-12+IL-21), in an amount of 0.5 μg each in combination with the CMV DNA vaccine construct (0.5 μg) (Table 2). Two weeks after the inoculation of the vaccine, the experimental animals were sacrificed and spleens were extracted and the T cell-specific immune response against CMV was analyzed by ex vivo ELISPOT analysis using CMVpp65 (i.e., an antigen of CMV included in the CMV DNA vaccine) and a CMVgB peptide pool (FIG. 8 a ). The CMVgB-specific antibody response was analyzed via the CMV-specific antibody response ELISA test method using obtained blood plasma (FIGS. 8 a to 8 c ).

TABLE 2

Compositions for administration of CMV
vaccine of the present disclosure

	No. of
	Heads for			Administration
Group	Experiment	Composition	Dose	Route

1	3	Mock	0.5 μg/	i. m. +
2	4	CMV DNA vaccine	plasmid	Electroporation
3	4	CMV DNA vaccine +
		IL-12
4	4	CMV DNA vaccine +
		IL-21
5	4	CMV DNA vaccine +
		IL-12 + IL-21
6	4	CMV DNA vaccine +
		BD121

As a result, as shown in FIG. 8 b , the BD-121 vaccine adjuvant according to an exemplary embodiment of the present invention resulted in a significantly higher T cell-specific immune response compared to when the CMV DNA vaccine was treated alone. Specifically, it was confirmed that when the CMV DNA vaccine was administered alone, the CMVpp65- and CMVgB-specific immune responses were 300 spot forming cells (SFCs) and 700 SFCs, respectively, whereas when the CMV DNA vaccine was administered in combination with BD-121, the CMVpp65- and CMVgB-specific immune responses were 2,000 SFCs and 2,100 SFCs, respectively, thus showing about 3-fold to 7-fold increase of antigen-specific T-cell responses. Meanwhile, as shown in FIG. 8 c , as a result of the analysis of the level of the CMV-specific antibody production, the BD-121 of the present invention induced a rather lower antibody response compared to when the CMV DNA vaccine was treated alone. These results suggest that the BD-121 according to an exemplary embodiment of the present invention induces the enhancement of immunity by selectively strengthening the T cell-specific immune response (i.e., cell-mediated immune response), instead of a method of non-selectively increasing an immune response.
3-2: T Cell-Specific Immune Response and Antibody Response Against HBV
The present inventors inoculated the HBV DNA vaccine construct (2 μg), which was prepared in Example 7, alone or in combination with BD-121 (2 μg), to the femoral muscles of C57BL/6 mice twice at two-week intervals using OrbiJector® (SLVAXiGEN, Korea) by in vivo electroporation (priming and boosting, respectively). On the 2^ndinoculation day, i.e., two weeks from the boosting, the experimental animals were sacrificed and spleens were extracted and the T cell-specific immune response against HBV was analyzed by ex vivo ELISPOT analysis using the HBV antigens (HBsAg, HBcAg, and PreS1/S2) peptide pool included in the HBV DNA vaccine construct (FIG. 8 a ). In addition, with regard to the HBsAg-specific antibody response, the HBV-specific antibody response was analyzed by ELISA test method using the obtained blood plasma (FIGS. 9 a and 9 b ).
As a result, as shown in FIG. 9 a , the BD-121 vaccine adjuvant according to an exemplary embodiment of the present invention resulted in a significantly higher T cell-specific immune response compared to when the HBV DNA vaccine construct was administered alone. In particular, the number of the spleen immune cells reactive to the HBsAg and the HBcAg antigens was drastically increased. However, as shown in FIG. 9 b , as a result of the analysis of the level of HBV-specific antibody production, it was confirmed that, contrary to the expectation, the BD-121 of the present invention did not increase the antibody titer compared to when the HBV DNA vaccine construct treated alone. These results confirm that selective strengthening of the T cell-specific immune response by the BD-121 of the present invention is not limited to a particular viral antigen, but it is a phenomenon that appears in a similar feature for most infectious viruses.
3-3: T Cell-Specific Immune Response and Antibody Response Against SFTS
The pGX-27 (G1) was administered as an empty vector, or the SFTS DNA vaccine (12 μg) was administered alone (G2) or in combination with BD-121 (12 μg) (G3) to the femoral muscles of C57BL/6 mice twice at two-week intervals using OrbiJector® (SLVAXiGEN, Korea) by in vivo electroporation (boosting). Then, some mice were sacrificed after the 1^stinoculation (priming) to extract spleens, and the T cell-specific immune response against the SFTS was then analyzed by the ELISPOT analysis using GnGc, NP, and NS, which are antigens of the SFTS included in the SFTS DNA vaccine, and the remaining experimental animals (n=5) were sacrificed two weeks after the 2^ndinoculation day (boosting) to extract spleens, and the T cell-specific immune response was analyzed via ELISPOT analysis as described above (FIGS. 10 a to 10 c ).
As a result, as shown in FIG. 10 b , the BD-121 vaccine adjuvant according to an exemplary embodiment of the present invention induced a significant T cell-specific immune response even based on priming alone, compared to when the SFTS DNA vaccine was treated alone, and as shown in FIG. 10 c , the T cell-specific immune response was further increased at the time of boosting. Meanwhile, when the SFTS DNA vaccine was administered alone, no enhancement of immune responses by boosting was observed. Accordingly, it was confirmed that the BD-121 according to an exemplary embodiment of the present invention exhibits a more effective vaccine adjuvant effect when boosted by the 2^ndinoculation as well as the priming by the 1^stinoculation.
Then, the present inventors compared the effects of priming and boosting by varying the inoculation rate of the SFTS DNA vaccine and the BD-121 vaccine adjuvant according to an exemplary embodiment of the present disclosure.
Specifically, an empty vector (mock DNA, GI), an SFTS DNA vaccine alone (G2), a vaccine where the amount of SFTS DNA vaccine:BD-121 is 1:0.3 (G3), a vaccine where the amount of SFTS DNA vaccine:BD-121 is 1:1 (G4), and a vaccine where the amount of SFTS DNA vaccine:BD-121 is 1:3 (G5) were administered to the femoral muscles of C57BL/6 mice twice at two-week intervals using OrbiJector® (SLVAXiGEN, Korea) by in vivo electroporation (boosting), respectively. Then, some mice were sacrificed after the 1^stinoculation (priming) to extract spleens, and the T cell-specific immune response against the SFTS was then analyzed by the ELISPOT analysis using GnGc, NP, and NS, which are antigens of the SFTS included in the SFTS DNA vaccine, and the remaining experimental animals (n=5) were sacrificed two weeks after the 2^ndinoculation (boosting) to extract spleens, and the SFTS-specific antibody response was analyzed via ELISPOT analysis by obtaining blood plasma (Table 3, FIGS. 11 a and 11 b ).

TABLE 3

Outline of BD-121 dose change experiment for SFTS DNA vaccine

	No. of
Experimental	Heads for	DNA Construct for	Administration
Group	Experiment	Administration	Route

G1
	3	Mock DNA	Intramuscular
G2
	5	SFTS DNA vaccine	Electroporation
G3
	5	SFTS DNA vaccine +
		BD-121 (1:0.3)
G4	5	SFTS DNA vaccine +
		BD-121 (1:1)
G5	5	SFTS DNA vaccine +
		BD-121 (1:3)

As a result, as shown in FIG. 11 a , there was no significant change caused by priming between the group administered with the SFTS DNA vaccine alone and the group administered with a vaccine (where the SFTS DNA vaccine and the BD-121 according to an exemplary embodiment of the present invention were mixed in a ratio of 1:0.3). However, the experimental group where the dose ratio was increased to 1:1 showed a distinct increase in the effect of the T cell-specific immune response. Meanwhile, in the case of boosting, as shown in FIG. 11 b , it was confirmed that there was a significant increase of the T cell-specific immune response compared to the group administered with SFTS alone, even when the dose ratio was 1:0.3. These results suggest that the BD-121 vaccine adjuvant can improve the T cell-specific immune response in a dose-dependent manner.
3-4: T Cell-Specific Immune Response Against HSV-2
The present inventors examined whether the BD-121A prepared according to an exemplary embodiment of the present invention can enhance the T cell-specific immune response when the HSV-2 DNA vaccine construct prepared in Example 8 was administered.
Specifically, the experimental animals were divided into a group administered with an empty vector (pGX-27) (n=10); a group administered with the BD-02B (0.49 μg) and the BD-02C (0.49 μg) prepared in Example 8 (n=11); a group administered with the BD-02B (1.4 μg) and the BD-02C (1.4 μg) prepared in Example 8 (n=9); a group administered with the BD-02B (0.49 μg), the BD-02C (0.49 μg), and the BD121A (0.49 μg) (n=11); and a group administered with the BD-02B (1.4 μg), the BD-02C (1.4 μg), and the BD121A (1.4 μg) prepared in Example 8 (n=10), and were administered to the femoral muscles of C57BL/6 mice twice at two-week intervals using OrbiJector® (SLVAXiGEN, Korea) by in vivo electroporation (priming and boosting)). Then, after one week of the boosting, Depo-provera (2 mg) was administered to adjust the sexual hormone cycle between female mice, and infected with HSV-2 two weeks after the boosting, and the pathological scores and survival rates of the experimental animals over time were then analyzed (FIGS. 12 a to 12 c ).
As a result, as shown in FIG. 12 b , the pathological scores of the experimental group, in which the BD121A according to an exemplary embodiment of the present invention was co-administered, were lower than those in the group in which the BD-02 was administered alone, and such a phenomenon was shown to be dose-dependent. Furthermore, as a result of the analysis of survival rates, as shown in FIG. 12 c , the group in which the BD-02 was administered alone showed an increase in the survival rates in a dose-dependent manner, compared to the control group, whereas the group (BD02 DP) in which the BD-121A according to an exemplary embodiment of the present invention was co-administered, the survival rates of experimental animals increased significantly. In particular, in the group where the BD-02 and BD-121A (BD02 DP) were administered in an amount of 1.4 μg, respectively, no dead animal was observed even after 18 days of infection.
Then, to confirm the optimal mixing ratio between BD-02 and BD-121A, the present inventors adjusted the BD-02 and the BD-121A at various ratios (BD-02B:BD-02C:BD121A=2:2:0.2, 2:2:0.6, 2:2:2, and n=5+5 per group) and inoculated into the femoral muscle of C57BL/6 mice twice at two-week intervals using OrbiJector® (SLVAXiGEN, Korea) by in vivo electroporation (priming and boosting). Then, 5 mice in each group were sacrificed two weeks after the priming while the remaining 5 mice in each group were sacrificed two weeks after the boosting. Then, spleens were extracted therefrom and the number of spleen immune cells reactive to various HSV-2 antigens included in the BD-02 was counted by the ELISPOT analysis (Table 4, and FIGS. 13 a and 13 b ).

TABLE 4

Outline of BD-121 dose change experiment
for HSV-2 DNA vaccine

Experimental	No. of	DNA construct for	Administration
Group	Heads	Administration	Route

1	5 + 5	BD-121A (6 μg)	Intramuscular
			Electroporation

2	5 + 5	BD-02B (2 μg) + BD-02C (2	Intramuscular
		μg) + BD-121A (0.2 μg)	Electroporation
3	5 + 5	BD-02B (2 μg) + BD-02C (2	Intramuscular
		μg) + BD-121A (0.6 μg)	Electroporation
4	5 + 5	BD-02B (2 μg) + BD-02C (2	Intramuscular
		μg) + BD-121A (2 μg)	Electroporation

As a result, as shown in FIG. 13 b , in the experimental group (BD-02B+BD-02C) where the BD-02 was administered alone, no T cell-specific immune response was observed two weeks after the priming. Meanwhile, in the group (BD02 DP) where the BD121A according to an exemplary embodiment of the present invention was co-administered, the T cell-specific immune response in a BD121A concentration-dependent manner was confirmed up to the ratio of 1:1:0.6. At higher doses, however, no further increase in the T cell-specific immune response was observed. Accordingly, in the BD-02 and BD121A vaccine composition according to an exemplary embodiment of the present disclosure, it was confirmed that a significant immunostimulating effect was shown even at a mixing ratio (BD-02B:BD-02C:BD121A=2:2:0.6).
3-5: Analysis of T Cell-Specific Immune Response Against HPV
The present inventors examined whether the BD-121 can enhance a T cell-specific immune response using DNA vaccine constructs of 2-valent HPV types 16 and 18 described in KR Patent Laid-open Publication No. 10-2017-0045254.
Specifically, C57BL/6 mice (experimental animals) were divided into a group administered with an empty vector (administration of pGX-27, n=10), a group administered with the 2-valent HPV DNA vaccine construct alone (n=10), a group administered with the 2-valent HPV DNA vaccine construct (8 μg)+BD-121 (1 μg) (n=10), and a group administered with the 2-valent HPV DNA vaccine construct (8 μg)+BD-121 (3 μg) (n=10). Then, two weeks after the 1^stvaccine administration (priming), a half of the experimental animals (per each experimental group, n=5) were sacrificed and the spleens were extracted and used for the analysis of the T cell-specific immune response according to the priming while the remaining half (per each experimental group, n=5) were subjected to the 2^ndinoculation (boosting) two weeks after the priming, and likewise, the remaining half were sacrificed two weeks after the boosting and the spleens were extracted and the T cell-specific immune response was analyzed by a method of counting the number of spleen immune cells which are specifically reactive to antigens by the ELISPOT analysis (FIG. 14 ).
As a result, as shown in FIG. 14 , the T cell-specific immune response was negligible when the 2-valent HPV DNA vaccine construct was administered alone, whereas when the BD-121 according to an exemplary embodiment of the present invention was administered, the T cell-specific immune response which is concentration-dependent was enhanced.
Then, the present inventors, in order to confirm the performance of the BD-121 and the BD-121A, as vaccine adjuvants, for the HPV 14 DNA vaccine (BD-14A) which was prepared according to an exemplary embodiment of the present disclosure, administered the BD-14s (BD-14A, BD-14B, and BD-14C), which were prepared in Example 5, alone or in combination with BD-121A and then analyzed the results (Table 5.

TABLE 5

Outline of effect analysis experiment of various vaccine
adjuvants against 14-valent HPV DNA vaccine

Experimental	DNA construct for	Administration	Administration
Group	Administration	Dose	Route

1	Mock	—	Intermuscular
2	BD-14	2 μg	Electroporation
3	BD-14 + Mip-1α	2 μg + 2 μg
4	BD-14 + IL-12/IL-21	2 μg + 2 μg
5	BD-14 + IL-12/IL-	2 μg + 2 μg +
	21 + Mip-1α	2 μg

Specifically, C57BL/6 mice (i.e., experimental animals) were divided into a group administered with an empty vector (n=10), a group administered with BD-14 (2 μg) (n=10), a group administered with BD-14 (2 μg)+MIP-1α (2 μg) (n=10), a group administered with BD-14 (2 μg)+BD-121 (2 μg) (n=10), and a group administered with BD-14 (2 μg)+BD-121 (2 μg)+Mip-1α (2 μg) (n=10). Then, two weeks after the 1^stvaccine administration (priming), a half of the experimental animals (per each experimental group, n=5) were sacrificed and the spleens were extracted and used for the analysis of the T cell-specific immune response according to the priming while the remaining half (per each experimental group, n=5) were subjected to the 2^ndinoculation (boosting) two weeks after the priming, and likewise, the remaining half of the experimental animals were sacrificed two weeks after the boosting and the spleens were extracted and the T cell-specific immune response was then analyzed by a method of counting the number of spleen immune cells which are specifically reactive to antigens by the ELISPOT analysis (FIGS. 15 a and 15 b ).
As a result, as shown in FIG. 15 b , both BD-121 and BD-121A significantly enhanced the T cell-specific immune response compared to the group administered with BD-10A alone and the group co-administered with BD-10A and MIP-1α.

Experimental Example 4: Preclinical Analysis

From the results of Example 3, the present inventors confirmed that the BD-121 according to an exemplary embodiment of the present invention can enhance a T cell-specific immune response against various virus antigens without much effect on antibody production. In this regard, the present inventors examined whether the BD-121 can alleviate pathological symptoms due to virus infection compared to the administration of a DNA vaccine alone, even in preclinical studies using a real animal model.
Specifically, as experimental animals, three cynomolgus monkeys were used per each experimental group, and the administration groups were divided into a group administered with BD-02 alone and a group co-administered with BD-121A. Each group was administered twice at intervals of three weeks with 1.8 mg of DNA via the muscular route by electroporation, and evaluation of immune responses specific for BD-02 was performed at a time point before the administration (VS, day −14 and day −5), a time point after the 1^stadministration (VT1, day 21), and a time point after the 2^ndadministration (VT2, day 35) (FIG. 16 a ). At each time point, the separated peripheral blood mononuclear cells (PBMCs) were stimulated with a peptide pool for HSV-2 gD and UL39 antigens, which are antigens included in BD-02, and those cells which exhibited specific responses were evaluated via the ex vivo ELIPOST analysis method and indicated them as spot forming cells (SFCs).
As a result, as shown in FIG. 16 b , it was confirmed that the BD-02-specific immune response was not observed at the time point before the administration, but the BD-02-specific immune response was increased after the administration in a dose frequency dependent manner. In particular, it was confirmed that the BD-02-specific immune response was significantly increased by the co-administration with BD-121A compared to the group administered with BD-02 alone, and it was observed that the difference became greater with the increase/decrease of the administration frequency.
As described above, it was confirmed that the BD-121 and the BD-121A according to an exemplary embodiment of the present invention can drastically enhance T cell-specific immune responses against various virus antigens from various virus infections, without much effect of antibody production. Accordingly, since the BD-121 and the BD-121A according to an exemplary embodiment of the present invention can drastically enhance T cell-specific immune responses which selectively increases T cells that react specifically to antigens without affecting antibody production, the BD-121 and the BD-121A according to an exemplary embodiment of the present invention can be used as an immunotherapeutic agent via cancer cell-mediated immunity enhancement for cancers (e.g., cervical cancer) as well as prevention and treatment of various infectious viruses. For example, in the case of antitumor immunotherapy responses, antibody responses is known to have little therapeutic effect on tumor cells or tumor-associated antigens, it is very important to effectively increase T cell responses specific for tumor cells or tumor-associated antigens. Even in the therapeutic immune response for the infections of viruses, such as HSV-2 or HPV, it is very important to effectively increase a T cell response because the T cell response, not the antibody response, plays a crucial role. For these reasons, the application of the BD-121 and the BD-121A, which can selectively improve only T cell responses that are specific for antigens or vaccines, has an advantage in that it can be led to the development of novel therapeutic agents for various diseases mentioned above.
Although a novel vaccine adjuvant has been described with reference to the specific embodiments, they are for illustrative purposes only. Therefore, it will be understood by those skilled in the art that various modifications and changes can be made thereto without departing from the spirit and scope of the present invention defined by the appended claims. Hence, the real protective scope of the present invention shall be determined by the technical scope of the accompanying claims.

INDUSTRIAL APPLICABILITY

The immune adjuvant according to an embodiment of the present invention may be usefully used as an immunotherapeutic agent through cellular immune enhancement of cancer, such as cervical cancer, as well as prevention and treatment of infection by various infectious viruses.

SEQUENCE LISTING FREE TEXT

SEQ ID NO: 1 is the amino acid sequence of human IL-12p35 polypeptide.
SEQ ID NO: 2 is the amino acid sequence of a human IL-12p40 polypeptide.
SEQ ID NO: 3 is the amino acid sequence of a human IL-21 polypeptide.
SEQ ID NO: 4 is a nucleic acid sequence of a polynucleotide encoding the human IL-12p35 polypeptide.
SEQ ID NO: 5 is a nucleic acid sequence of a polynucleotide encoding the human IL-12p40 polypeptide.
SEQ ID NO: 6 is the nucleic acid sequence of the EMCV-derived internal ribosome entry site.
SEQ ID NO: 7 is the nucleic acid sequence of the RSV promoter.
SEQ ID NO: 8 is a nucleic acid sequence of a polynucleotide encoding the human IL-12 polypeptide.
SEQ ID NO: 9 is the nucleic acid sequence of the human EF-1α promoter.
SEQ ID NO: 10 is the amino acid sequence of a human MIP-1α polypeptide.
SEQ ID NO: 11 is a nucleic acid sequence of a polynucleotide encoding the human MIP-1α polypeptide.
SEQ ID NO: 12 is the amino acid sequence of the mouse IL-12p35 polypeptide.
SEQ ID NO: 13 is the amino acid sequence of a mouse IL-12p40 polypeptide.
SEQ ID NO: 14 is a nucleic acid sequence of a polynucleotide encoding the mouse IL-12p35 polypeptide.
SEQ ID NO: 15 is a nucleic acid sequence of a polynucleotide encoding the mouse IL-12p40 polypeptide.
SEQ ID NO: 16 is the amino acid sequence of a mouse IL-21 polypeptide.
SEQ ID NO: 17 is a nucleic acid sequence of a polynucleotide encoding the mouse IL-21 polypeptide.
SEQ ID NO: 18 is the amino acid sequence of a mouse MIP-1α polypeptide.
SEQ ID NO: 19 is a nucleic acid sequence of a polynucleotide encoding the mouse MIP-1α polypeptide.
SEQ ID NO: 20 is the amino acid sequence of the (GS)5 linker peptide.
SEQ ID NO: 21 is the nucleic acid sequence of a polynucleotide encoding the (GS)₅linker peptide.
SEQ ID NO: 22 is the amino acid sequence of the tPA leader peptide.
SEQ ID NO: 23 is a nucleic acid sequence of a polynucleotide encoding the tPA leader peptide.
SEQ ID NO: 24 is the amino acid sequence of the Flt3L polypeptide whose signal sequence is truncated.
SEQ ID NO: 25 is a nucleic acid sequence of a polynucleotide encoding the Flt3L polypeptide.
SEQ ID NOs: 26, 28 and 30 are amino acid sequences of shuffled antigen fusion proteins included in a DNA vaccine construct according to an embodiment of the present invention, respectively.
SEQ ID NOs: 27, 29, and 31 are nucleic acid sequences of polynucleotides encoding the shuffled antigen complexes, respectively.
SEQ ID NO: 32 is the amino acid sequence of the UL39-N1 polypeptide.
SEQ ID NO: 33 is the amino acid sequence of the UL39-C2 polypeptide.
SEQ ID NO: 34 is the amino acid sequence of the UL39-N2 polypeptide.
SEQ ID NO: 35 is the amino acid sequence of the UL39-N4-C1 polypeptide.
SEQ ID NO: 36 is the amino acid sequence of the UL39-N3 polypeptide.
SEQ ID NO: 37 is the amino acid sequence of the shuffled UL39 antigen protein sequentially linked by UL39-N1, UL39-C2, UL39-N2, UL39-N4-C1, and UL39-N3.
SEQ ID NO: 39 is the amino acid sequence of the HSV-2 glycoprotein D (gD) with the signal sequence and transmembrane domain removed.
SEQ ID NO: 40 is the amino acid sequence of the ICP0 protein from which the nuclear positioning sequence has been removed.
SEQ ID NO: 41 is the amino acid sequence of the ICP4 protein from which the RS1.3 portion has been removed.
SEQ ID NO: 42 is the amino acid sequence of the tPA-Flt3L-gD-ICP0-ICP4 fusion protein.
SEQ ID NO: 43 is a nucleic acid sequence of a polynucleotide encoding the fusion protein of SEQ ID NO: 42.
SEQ ID NO: 44 is the amino acid sequence of the antigen GnGc protein of SFTS.
SEQ ID NO: 45 is the amino acid sequence of the NP protein of SFTS.
SEQ ID NO: 46 is the amino acid sequence of the NS protein of SFTS.
SEQ ID NOs: 47 to 49 are nucleic acid sequences of polynucleotides encoding the GnGc, NP and NS proteins, respectively.
SEQ ID NOs: 50 to 68 are amino acid sequences of various linker peptides that can be used in the present invention.

Claims

1.-16. (canceled)

17. A method for stimulating a T lymphocyte-specific immune response by a vaccine composition in a subject comprising administering an adjuvant composition comprising i) IL-12 protein and IL-21 protein; or ii) a polynucleotide encoding a p35 chain of IL-12 protein (IL-12p35), a polynucleotide encoding a p40 chain of IL-12 protein (IL-12p40) and a polynucleotide encoding an IL-21 protein along with the vaccine composition to the subject,

wherein the adjuvant composition induces a lower antibody response compared to when the vaccine composition is treated alone.

18. The method of claim 17, wherein each of the polynucleotides is operably linked to a promoter or

an internal ribosome entry site (IRES) links between at least two of the polynucleotides and the polynucleotides including the IRES is operably linked to a promoter.

19. The method of claim 17, wherein the adjuvant composition comprises one to three vectors, each of which comprises: polynucleotides that respectively encode the IL-12p35 and the IL-12p40; and a polynucleotide that encodes the IL-21 protein; or mRNA molecules, each of which encodes the IL-12p35, IL-12p40, and IL-21 proteins.

20. The method of claim 17, wherein the IL-12p35 is a human IL-12p35 consisting of the amino acid sequence of SEQ ID NO: 1.

21. The method of claim 17, wherein the IL-12p40 is a human IL-12p40 consisting of the amino acid sequence of SEQ ID NO: 2.

22. The method of claim 17, wherein the IL-21 protein is a human IL-21 consisting of the amino acid sequence of SEQ ID NO: 3.

23. The method of claim 17, the adjuvant composition further comprises at least one among:

a MIP-1α gene construct, in which a polynucleotide encoding the MIP-1α protein is operably linked to a promoter;

a complex gene construct, in which a polynucleotide encoding the MIP-1α protein is operably linked to at least one among the IL-12p35, IL-12p40, and IL-21 proteins, by a polynucleotide encoding an internal ribosome entry site (IRES) or linker peptide; and

a mRNA molecule encoding MIP-1α protein.

24. The method of claim 23, wherein the MIP-1α protein consists of the amino acid sequence of SEQ ID NO: 10.

25. The method claim 23, wherein the MIP-1α gene construct is comprised in a separate expression vector or comprised in any one or more of the one to three vectors, each of which comprises: polynucleotides that respectively encode a p35 chain (IL-12p35) and a p40 chain (IL-12p40) that constitute the IL-12 protein; and a polynucleotide that encodes the IL-21 protein.

26. The method of claim 17, wherein the vaccine composition comprises at least one antigen derived from infectious virus or at least one cancer antigen or a polynucleotide encoding the antigen derived from infectious virus or the cancer antigen.

27. The method of claim 26, wherein the infectious virus is Epstein-Barr virus (EBV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), Hantaan virus, cytomegalovirus (CMV), human immunodeficiency virus (HIV), influenza virus, human papillomavirus (HPV), poliovirus, ebola virus, rotavirus, dengue virus, West Nile virus, yellow fever virus, adenovirus, Japanese encephalitis virus, BK virus, smallpox virus, Zika virus, severe fever with thrombocytopenia syndrome (SFTS) virus, or herpes simplex virus (HSV).

28. The method of claim 26, wherein the cancer antigen is a human papillomavirus (HPV)-derived antigen, a carcinoembryonic antigen, a prostate-specific membrane antigen (PSMA), Her2/neu, MUC-1, BCR/ABL, α-fetoprotein (AFP), an Epstein-Barr virus (EBV)-derived antigen, a human hepatitis B virus (HBV)-derived antigen, a human hepatitis C virus (HCV)-derived antigen, cancer antigen-125 (CA-125), cancer antigen-72-4 (CA-72-4), cancer antigen-15-3 (CA-15-3), or cancer antigen-19-9 (CA-19-9).

29. The method of claim 17, wherein the adjuvant composition is administrated via intramuscular injection, intravenous injection or electroporation.